Relevance to R21 RFA: Our application will characterize PmrA, the quinolone efflux pump of S. pneumoniae that is overexpressed in response to hydrophilic quinolones. We believe that this overexpression leads to an unnecessarily high rate of resistance. This application is novel since it focuses on the role of efflux pumps in predisposing to subsequent mutations in topoisomerase genes rather than primary mutations in the topoisomerase genes themselves. We believe that mutations in S. pneumoniae causing constitutive overexpression of its efflux pump [ PmrA], as well induction causing efflux pump upregulation in WT cells, predispose to topoisomerase mutations. Notably, the chemicals that cause quinolone pump induction in other bacteria include quinolones themselves. Global regulation in response to environmental conditions may also upregulate PmrA leading to resistance. Specific Aim #1 will characterize mutational PmrA upregulation as well as pump induction in WT cells and the effects this has on quinolone susceptibility. Preliminary studies will identify chemicals that are pump substrates, which are also potential inducers, by comparing their MICs in the presence and absence of reserpine. To survey these potential inducers we will use a pmrAlacZ reporter in a a-galactosidase negative strain of S. pneumoniae, mRNA and protein will be measured in the presence of inducers in WT strains and in strains that do not express pmrA as well as mutants with constitutive expression by Real Time PCR and Western blots. Specific Aim #2 will characterize the efflux kinetics of quinolones with different hydrophilicities using everted membrane vesicles of E. coli that express the PmrA efflux pump of S. pneumoniae. In these studies we will use radiolabeled quinolones, uptake measured by membrane filtrations, the Vmax and Km will be determined. Long term objectives: We expect that the new information we provide will lead to more rational approaches to quinolone therapy considering ways to prevent efflux pump mutant selection or upregulation in WT cells minimizing subsequent topoisomerase mutant selection. Using everted vesicles in conjunction with kinetic data from other subcellular systems describing kinetic parameters describing uptake and topoisomerase interactions, ultimately will provide a model to quantify the relative roles each has on resistance as well as develop a novel, mechanism related screen that may be useful in developing new quinolones with enhanced activity against resistant isolates.